Journal of Ethnopharmacology 91 (2004) 43–50

Acute and chronic toxicological studies of

Ajuga iva in experimental animals
Jaouad El Hilaly a , Zafar H. Israili b , Badiâa Lyoussi a,∗
a UFR Physiology-Pharmacology, Laboratory of Animal Physiology, Department of Biology,
Faculty of Sciences Dhar El Mehraz, BP 1976 Atlas, Fez, Morocco
b Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA

Received 8 October 2003; received in revised form 21 October 2003; accepted 17 November 2003

Abstract

Ajuga iva (L.) Schreber (AI), is widely used in the Moroccan pharmacopoeia as a panacea (cure-all), and specifically for gastrointestinal
disorders and diabetes, and as an anthelmintic. No toxicological investigations have been carried out on this plant. We have previously
observed that single oral doses (2–14 g/kg) of a lyophilised aqueous extract of AI (AI-extract) in mice or daily oral administration of
10 mg/kg of AI-extract in rats for 2 weeks did not result in any adverse effects. We have now evaluated AI-extract for its behavioural and
pharmaco-toxicological effects after acute and chronic administration by the oral and intraperitoneal routes in rats and mice. No toxicity was
observed in mice after single oral doses of as high as 14 g/kg of the AI-extract. However, single intraperitoneal injections of the AI-extract
(1500–5500 mg/kg BW) produced a dose-dependent increase in adverse effects in the general behaviour and the mortality rate; the LD50 of
acute intraperitoneal dose was 3.6 g/kg. In chronic toxicological studies in rats, the AI-extract (administered orally at daily doses of 100, 300
and 600 mg/kg for 3 months), did not cause any changes in haematological and biochemical parameters, with the exception of a transient rise
in platelet counts and a short-term decrease in serum glucose levels. Histopathological examination of the brain, liver and the kidneys at the
end of the study (3 months) showed normal architecture suggesting no morphological disturbances.
© 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Ajuga iva; Folk medicine; Animal toxicology

1. Introduction herbaceous plant, commonly found in different regions of

Several species of the genus Ajuga (Labiatea) are used in
African and Asian folk medicine. In the Moroccan pharma-
copoeia, Ajuga iva (L.) Schreber (AI) is known for its numer-
ous beneficial effects as a panacea (cure-all) (Hassar, 1999),
and specifically for gastrointestinal disorders (Bellakhdar
et al., 1991), hypertension (Ziyyat et al., 1997) and dia-
betes (Ziyyat et al., 1997), and as an anthelmintic. In East
Africa, the plants of the genus Ajuga have been used as a
remedy for fever, toothache, dysentery, and high blood pres-
sure (Kokwaro, 1976), and in the traditional Chinese phar-
macopoeia, they are known for their diuretic effect (Aliotta
and Pollio, 1994). It is worthy of note that AI is an annual
Abbreviations: AI, Ajuga iva; ALT, alanine aminotransferase; AST,
aspartate aminotransferase; EDTA, ethylenediamine tetraacetate; LOAEL,
lowest-observed-adverse-effect level; NOAEL, no-observed-adverse-effect
level; RBC, red blood cells
∗ Corresponding author.
E-mail address: lyoussi@rocketmail.com (B. Lyoussi).
Morocco, where it is known as “Chendgora” (Ziyyat et al.,
1997). According to our survey carried out in North Mo-
rocco, AI powder is also smoked by some tribes (El-Hilaly
et al., 2003).
Importantly, the plants of the genus Ajuga, which are eco-
logically related, have been shown to display a wide spec-
trum of biological and pharmacological activities, which
provide experimental support for the empiric ethnophar-
macological use of this plant in folk medicine. The plants
of the genus Ajuga have been reported to have antifungal
(Anon, 2000; Kariba, 2001), antibacterial (Chen et al., 1996;
Anon, 2000; Bennaghmouch et al., 2001), antimycobacte-
rial (Cantrell et al., 1999), antihypertensive (Odek-Ogunde
et al., 1993), antiplasmodial (Kuria et al., 2001, 2002),
hypoglycaemic (Hilaly and Lyoussi, 2002), and larvae and
insect antifeedant (Bremner et al., 1998; Bondı̀ et al., 2000;
Ben Jannet et al., 2000, 2001) activity. The empirical use of
Ajuga is corroborated by the isolation and identification of
a number of the active compounds, including antileukemic
sterol glycosides (Akbay et al., 2002), hypoglycaemic

0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2003.11.009
44 J.E. Hilaly et al. / Journal of Ethnopharmacology 91 (2004) 43–50

ecdysteroids (Kutepova et al., 2001), antibacterial and insect
antifeedant neo-clerodane diterpenoids (Chen et al., 1996;
Bremner et al., 1998; Ben Jannet et al., 2000; Bondı̀ et al.,
2000), insect antifeedant diglyceride (Ben Jannet et al.,
2001), vasoconstrictor 8-O-acetyharpagide (Breschi et al.,
1992), and insect ecdysis inhibitors (Kubo et al., 1981).
Some of the phytochemicals isolated from the Ajuga plants
are reported to be cardiotonic (Kuria and Muriuki, 1984),
renal stimulant (Aliotta and Pollio, 1994), biliary sectreta-
gogue (Syrov et al., 1986), antidote for liver toxicity (Syrov
and Khushbaktova, 2001), and erythropoiesis stimulator
(Syrov et al., 1997). In addition, the flavones isolated from
Ajuga decumbens are potent inhibitors of human immunode-
ficiency virus reverse transcriptase (Tang et al., 1994), while
other compounds (e.g. cyasterone and 8-acetylharpagide)
isolated from the same species, show strong inhibitory ef-
fect on the induction of Epstein-Barr virus early antigen
(Takasaki et al., 1998, 1999) and antitumor-promoting ac-
tivity in two-stage models of carcinogenesis (Takasaki et al.,
1999; Konoshima et al., 2000).
Notwithstanding the widespread use of Ajuga plants in
traditional medicine and despite the fact that many plants
exhibit significant toxicity, some as serious as enhance-
ment of mutagenecity, carcinogenecity or embryotoxicity,
no systematic toxicological study has been undertaken in
any species of this genus. Therefore, the aim of the present
study was to carry out basic toxicological studies and estab-
lish the safety of an aqueous extract of the whole AI plant
(AI-extract), focusing on its acute and chronic toxicity in
rats and mice.
2. Material and methods
2.1. Plant material
Mature whole AI plants were collected in North Morocco
between April and May 1998 and stored at room temperature
in a dry place prior to use. The plants were authenticated as
Ajuga iva L. by Professor M. Fennan of the Department of
Botany, Scientific National Institute (Rabat), and a voucher
specimen (H63) was deposited in the Institute.
2.2. Preparation of the aqueous extract of Ajuga iva
The whole AI plant was washed in running water, then
dried and ground to a powder. The AI-extract was prepared
as follows: the powder was suspended in distilled water (50 g
powder per 500 ml water) and the mixture was heated un-
der reflux for 30 min, after which the decoction was cen-
trifuged and filtered. The filtrate was frozen at −20 ◦ C and
then lyophilised (FreeZone® Dry 4.5, USA). The crude yield
of the lyophilised material (AI-extract) was approximately
25% (w/w); it was stored at −20 ◦ C until further use.
2.3. Acute toxicity studies in mice
Healthy IOPS OFA mice of either sex, weighing 18–33 g,
were divided in groups of 10. The animals were housed three
per plastic cage, and the photoperiod (light on from 06:00
to 18:00 h), air changes and room temperature (24 ± 1 ◦ C)
were controlled. All animals had free access to tap water and

Table 1
Acute toxicity of a lyophilised aqueous extract of Ajuga iva administered by intraperitoneal injection to mice
Dose of AI-extract (mg/kg)a Sex D/T Mortality latency (h) Toxic symptoms

0 Male 0/5 – None

Female 0/5 – None
1500 Male 0/5 – None
Female 0/5 – None
2000 Male 0/5 – Hypoactivity, piloerection
Female 1/5 >48, <60 Hypoactivity, piloerection
2500 Male 1/5 >36, <48 Anorexia, hypoactivity, piloerection
Female 1/5 >36, <48 Anorexia, hypoactivity, piloerection
3000 Male 1/5 >36, <48 Anorexia, hypoactivity, salivation, asthenia
Female 2/5 >36, <48 Anorexia, hypoactivity, salivation, asthenia
3500 Male 2/5 >36, <48 Asthenia, anorexia, salivation, diarrhoea
Male 2/5 >36, <48 Asthenia, anorexia, salivation, diarrhoea
4000 Male 2/5 >36, <48 Asthenia, anorexia, salivation, diarrhoea, syncope
Female 4/5 >36, <48 Asthenia, anorexia, salivation, diarrhoea, syncope
4500 Male 3/5 >24, <36 Asthenia, anorexia, salivation, diarrhoea, syncope
Female 4/5 >24, <36 Asthenia, anorexia, salivation, diarrhoea, syncope
5000 Male 4/5 >24, <36 Asthenia, anorexia, salivation, diarrhoea, syncope
Female 5/5 >24, <36 Asthenia, anorexia, salivation, diarrhoea, syncope
5500 Male 5/5 >24, <36 Asthenia, anorexia, salivation, diarrhoea, syncope
Female 5/5 >24, <36 Asthenia, anorexia, salivation, diarrhoea, syncope
D/T = dead/treated mice; none = no toxic symptoms during the observation period; mortality latency = time to death (in days) after the injection.
a The lyophilised aqueous extract of Ajuga iva was dissolved in distilled water and administered as a single intraperitoneal dose to groups of mice.

Mice in each dose group (n = 10) were carefully examined for any signs of toxicity (behavioural changes and mortality) for 14 days.
 J.E. Hilaly et al. / Journal of Ethnopharmacology 91 (2004) 43–50 45

Table 2
Changes in the body weight of rats after chronic oral treatment with a lyophilised aqueous extract of Ajuga iva
DAY Group I (control) Group II Group III Group IV

D0 221.1 ± 28.3 209.2 ± 34.9 201.3 ± 31.3 196.8 ± 28.8

D15 232.7 ± 27.9∗ 239.9 ± 39.9∗∗ 220.3 ± 35.7∗ 225.4 ± 26.1∗
D30 245.5 ± 28.7∗ 254.1 ± 50.4 ∗∗ 237.2 ± 35.1∗ 234.1 ± 26.9∗
D45 255.2 ± 26.6∗ 260.3 ± 50.8∗∗∗ 245.0 ± 42.8∗ 240.3 ± 36.0∗
D60 246.1 ± 29.7∗ 247.3 ± 62.1∗ 237.0 ± 43.3∗ 241.3 ± 42.1∗
D75 245.8 ± 33.2∗ 241.2 ± 60.7∗ 231.6 ± 36.9∗ 224.3 ± 40.1∗
D90 244.5 ± 27.6∗ 247.8 ± 57.4∗ 235.7 ± 33.9∗ 231.7 ± 43.3∗
The aqueous extract of the whole plant was given daily by the oral route to groups of Wistar rats (n = 10) at the doses: I (0 mg/kg, control), II
(100 mg/kg), III (300 mg/kg) and IV (600 mg/kg) for 90 days. The rats were weighed every 15 days. The data are expressed as mean ± S.E.M.; significant
differences for each group at each time period vs. pre-treatment values (D0) are as follows: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. There were no
significant differences in the body weights of the rats between the groups for each time period.

food, except for a short fasting period before oral adminis-
tration of single doses of the AI-extract. The AI-extract was
dissolved/suspended in distilled water and administered by
gavage at doses of 0, 2, 4, 6, 10 and 14 g/kg or by the in-
traperitoneal route at doses of 0, 1500, 2000, 2500, 3000,
3500, 4000, 4500, 5000 and 5500 mg/kg. The general be-
haviour of mice was observed continuously for 1 h after the
treatment and then intermittently for 4 h, and thereafter over
a period of 24 h (Twaij et al., 1983). The mice were further
observed for up to 14 days following treatment for any signs
of toxicity and deaths, and the latency of death. The LD50
value was determined according to the method of Litchfield
and Wilcoxon (1949).
2.4. Chronic toxicity studies in rats
Wistar rats (240–360 g) were housed five in each cage,
under the same conditions as described above for the mice.
The animals were divided into four groups (I–IV) of 10 rats
each (5 females and 5 males). The AI-extract was dissolved
in distilled water and administered orally, daily for 90 days
to the Groups I–IV at doses of 0, 100, 300 and 600 mg/kg,

Table 3
Effect of chronic oral administration of a lyophilised aqueous extract of Ajuga iva on the haematological parameters of Wistar rats
Haematological parameters Period of treatment (days)

D0 D30 D60 D90

Group I (control)
RBC (106 ␮l−1 ) 8.06 ± 0.23 7.95 ± 0.21 8.27 ± 0.18 7.91 ± 0.19
WBC (103 ␮l−1 ) 11.63 ± 0.70 10.48 ± 0.86 10.95 ± 0.63 10.92 ± 1.05
Haemoglobin (g/dl) 14.78 ± 0.57 15.17 ± 0.32 15.57 ± 0.17 15.09 ± 0.17
Haematocrit (vol.%) 41.76 ± 0.65 41.58 ± 0.68 41.07 ± 0.39 41.08 ± 0.35
Platelets (104 ␮l−1 ) 67.52 ± 2.92 67.20 ± 2.66 71.37 ± 4.77 72.38 ± 5.12
Group II (dose: 100 mg/kg)
RBC (106 ␮l−1 ) 7.66 ± 0.22 7.72 ± 0.16 8.17 ± 0.13 7.77 ± 0.10
WBC (103 ␮l−1 ) 12.32 ± 0.94 10.62 ± 0.73 13.72 ± 0.77 11.13 ± 0.94
Haemoglobin (g/dl) 14.60 ± 0.50 15.01 ± 0.24 15.37 ± 0.18 14.81 ± 0.23
Haematocrit (vol.%) 40.11 ± 0.68 41.33 ± 0.81 39.68 ± 0.70 41.18 ± 0.89
Platelets (104 ␮l−1 ) 69.81 ± 2.13 73.56 ± 2.38∗∗ 75.35 ± 5.99 81.43 ± 4.41
Group III (dose: 300 mg/kg)
RBC (106 ␮l−1 ) 7.96 ± 0.40 7.94 ± 0.29 8.51 ± 0.20 8.13 ± 0.23
WBC (103 ␮l−1 ) 12.26 ± 0.86 11.48 ± 1.00 13.11 ± 0.92 11.48 ± 1.39
Haemoglobin (g/dl) 14.70 ± 0.36 15.18 ± 0.41 15.17 ± 0.48 15.05 ± 0.11
Haematocrit (vol.%) 41.07 ± 0.75 42.06 ± 0.97 40.84 ± 0.89 40.98 ± 0.36
Platelets (104 ␮l−1 ) 63.79 ± 4.04 63.54 ± 3.73∗ 78.44 ± 5.82∗ 79.14 ± 4.39
Group IV (dose: 600 mg/kg)
RBC (106 ␮l−1 ) 8.26 ± 0.34 7.78 ± 0.19 7.97 ± 0.17 7.96 ± 0.20
WBC (103 ␮l−1 ) 11.49 ± 0.77 11.03 ± 0.97 12.81 ± 1.23 11.17 ± 0.97
Haemoglobin (g/dl) 14.11 ± 0.47 15.04 ± 0.31 14.81 ± 0.31 14.52 ± 0.26
Haematocrit (vol.%) 40.25 ± 0.86 40.49 ± 0.98 39.65 ± 0.78 39.89 ± 0.52
Platelets (104 ␮l−1 ) 68.17 ± 2.29 77.03 ± 2.82∗∗ 89.68 ± 3.15 99.43 ± 5.37
The aqueous extract of the whole plant was given daily by the oral route to groups of rats (n = 10) at the doses: I (0 mg/kg, control), II (100 mg/kg),
III (300 mg/kg) and IV (600 mg/kg) for 90 days. Haematological parameters were measured every 30 days. The data are expressed as mean ± S.E.M.;
significant differences in each group vs. the controls were as follows: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
46 J.E. Hilaly et al. / Journal of Ethnopharmacology 91 (2004) 43–50

respectively. Toxic manifestations and mortality were mon-
itored daily, and the body weight changes were recorded
every 15 days. At the end of every 30-day period, blood
was obtained from the retro-orbital puncture (Waynforth,
1980) under light diethyl ether anaesthesia using capillary
tubes. Blood was collected in two type of tubes: one with
EDTA and the other without any additives. The anticoagu-
lated blood (EDTA) was analysed immediately for haema-
tological parameters. The second tube was centrifuged at
4000 rpm at 4 ◦ C for 10 min to obtain the serum, which was
stored at −20 ◦ C until analysis for biochemical parameters.
At the end of the 90-day period, the animals were sacri-
ficed by decapitation and selected organs (brain, liver and
kidney) were carefully dissected out. Portions of these or-
gans were fixed in 10% formalin for histopathological ex-
aminations.
transferase (ALT) (IFCC, 1980) were determined enzymati-
cally using specific kits and measurement of optical density
at the corresponding wavelength with a spectrophotometer
(Ciba Corning 550 Express, England).
2.6. Statistical analysis
Results are expressed as mean ± standard error of mean
(S.E.M.). Significance differences between control and
experimental groups were assessed by Student’s t-test.
P-values less than 0.05 were considered to be significant.
3. Results
3.1. Acute toxicity in mice

2.5. Blood analysis
The haematological parameters (total red blood cell
(RBC), leukocyte (WBC) and platelet counts, haematocrit,
and haemoglobin) were determined using an autoanalyzer
(System H1, Bayer Diagnostics). The biochemical pa-
rameters, serum creatinine (Jaffe, 2003), glucose (Neese
et al., 1976), cholesterol (Parekh and Jung, 1970), aspartate
aminotransferase (AST) (IFCC, 1975) and alanine amino-
There were no deaths or any signs of toxicity observed
after oral administration of single doses of the AI-extract
at any dose level up to the highest dose tested (14 g/kg),
which was the no-observed-adverse-effect level (NOAEL)
(Alexeeff et al., 2002). However, the mortality rate as well
as the acute toxicity of the intraperitoneally administered
AI-extract increased progressively with the increasing dose
(Table 1): the mortality rate of 0% at and up to a dose of
1500 mg/kg gradually rose to 100% at 5500 mg/kg, the high-

Table 4
Effect of chronic oral administration of a lyophilised aqueous extract of Ajuga iva on the biochemical parameters of Wistar rats
Biochemical parameters Period of treatment (days)

D0 D30 D60 D90

Group I (control)
Glucose (mg/dl) 87.8 ± 5.0 87.5 ± 5.4 87.9 ± 3.1 90.8 ± 4.4
Cholesterol (mg/dl) 60.1 ± 5.1 63.2 ± 3.2 61.0 ± 3.1 58.8 ± 4.3
Creatinine (mg/dl) 51.2 ± 3.2 52.2 ± 1.2 53.4 ± 1.6 53.7 ± 1.8
AST (units/l) 139.2 ± 7.0 139.6 ± 5.4 39.1 ± 5.7 40.0 ± 7.3
ALT (units/l) 46.8 ± 6.0 50.1 ± 8.2 49.2 ± 7.7 44.7 ± 4.3
Group II (dose: 100 mg/kg)
Glucose (mg/dl) 90.7 ± 2.4 73.9 ± 2.8∗ 77.2 ± 3.3∗ 77.7 ± 6.2
Cholesterol (mg/dl) 55.8 ± 3.8 56.9 ± 3.7 55.9 ± 4.4 63.2 ± 2.7
Creatinine (mg/dl) 53.4 ± 2.1 52.0 ± 2.3 50.6 ± 1.5 56.4 ± 2.4
AST (units/l) 135.7 ± 3.9 143.3 ± 8.7 142.1 ± 8.9 39.4 ± 6.1
ALT (units/l) 52.0 ± 4.1 50.7 ± 3.7 45.1 ± 6.1 48.8 ± 3.5
Group III (dose: 300 mg/kg)
Glucose (mg/dl) 85.3 ± 3.7 78.8 ± 6.5 77.8 ± 2.9∗ 80.2 ± 3.7
Cholesterol (mg/dl) 60.4 ± 3.8 53.0 ± 6.7 51.2 ± 4.7 62.2 ± 2.8
Creatinine (mg/dl) 50.1 ± 2.9 55.3 ± 1.7 53.0 ± 1.6 53.6 ± 1.6
AST (units/l) 136.3 ± 3.9 134.2 ± 2.7 133.3 ± 4.3 138.1 ± 6.6
ALT (units/l) 50.2 ± 3.1 50.8 ± 2.8 46.5 ± 4.00 49.0 ± 2.8
Group IV (dose: 600 mg/kg)
Glucose (mg/dl) 90.2 ± 3.5 71.6 ± 3.6∗ 78.5± 6.4 80.5 ± 3.9
Cholesterol (mg/dl) 62.4 ± 4.3 50.3 ± 7.0 52.1± 6.5 61.9 ± 2.8
Creatinine (mg/dl) 52.4 ± 1.6 53.3 ± 1.6 50.3 ± 2.0 52.6 ± 1.3
AST (units/l) 140.6 ± 7.0 139.9 ± 7.00 135.3 ± 5.7 131.4 ± 6.3
ALT (units/l) 49.1 ± 3.8 51.2 ± 2.6 48.7 ± 3.0 44.8 ± 4.1
The aqueous extract of the whole plant was given daily by the oral route to groups of rats (n = 10) at the doses: I (0 mg/kg, control), II (100 mg/kg),
III (300 mg/kg) and IV (600 mg/kg) for 90 days. Biochemical parameters were measured every 30 days. The data are expressed as mean ± S.E.M.;
significant differences in each group vs. the controls were as follows: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.
 J.E. Hilaly et al. / Journal of Ethnopharmacology 91 (2004) 43–50
est dose studied. The NOAEL for the intraperitoneal dose
was 1500 mg/kg, while the lowest-observed-adverse-effect
level (LOAEL) was 2000 mg/kg. Some adverse effects, such
as hypoactivity and salivation, were seen immediately af-
ter the intraperitoneal injection, while others (e.g. anorexia
and weight loss) were observed later, and were more pro-
nounced at the higher doses and persisted until death. The
acute intraperitoneal toxicity (LD50 ) of AI-extract in mice
was 3600 mg/kg.
3.2. Chronic toxicity studies in rats
3.2.1. Effect of chronic oral administration of AI-extract
on the body weight and mortality
The body weights of control and AI-extract-treated rats
are presented in Table 2. No significant difference in body
weight gain was noted between the control and any of the
treated groups at any time period. Moreover, no lethality
was recorded for any dose up to the maximum of 600 mg/kg
(NOAEL) during the 90 days of treatment.
3.2.2. Effect of chronic oral administration of AI-extract
on the haematological and biochemical parameters of rats
The effect of chronic oral administration of AI-extract on
the haematological parameters is presented in Table 3. Only
the platelet counts were slightly but significantly higher in
the treated groups on the 30th day of treatment as com-
pared to the controls (Group II versus Group I, P < 0.05).
This increase was blunted at the end of the treatment (90th
day) at each dose level. All other parameters (haemoglobin,
haematocrit, RBC and WBC) remained within normal limits
throughout the treatment period.
The biochemical profiles of the treated and control rats
are presented in Table 4. Chronic oral administration of
AI-extract (up to a dose of 600 mg/kg BW) did not cause
significant changes in serum creatinine, cholesterol and the
activity of the marker enzymes (ALT, AST). However, blood
glucose level was significantly decreased in treated animals
(P < 0.05) as compared to the controls up to 60 days of
treatment; the hypoglycaemic effect was attenuated at the
end of the treatment period (90 days).
4. Discussion
Although, poisonous plants are ubiquitous (Kingsbury,
1964), herbal medicine is used by up to 80% of the popu-
lation in the developing countries. Despite the widespread
use, few scientific studies have been undertaken to ascertain
the safety and efficacy of traditional remedies. The present
investigation shows that an aqueous extract of the whole
plant Ajuga iva, is non-toxic via the oral route in mice and
rats, at least up to the maximum doses (NOAEL; Alexeeff
et al., 2002) of 14 and 600 mg/kg, respectively. Since the
AI-extract has previously been shown to be pharmacologi-
cally active (hypoglycaemic) when given by the oral route
47
to rats (Hilaly and Lyoussi, 2002) at the minimum active
dose of 10 mg/kg, one may conclude that the active com-
pound(s) present in the AI-extract exhibit a rather low acute
oral toxicity profile. In the present study, mortality and symp-
toms of pronounced adverse behaviour were noted only af-
ter the intraperitoneal injection of relatively high doses of
the AI-extract (LD50 = 3.6 g/kg) in mice.
The component(s) of the whole plant extract responsible
for the toxic manifestations after the intraperitoneal dose are
not known. The toxicity and the lethality of the AI-extract
may be due to any one or more of the phytochemicals present
in the crude aqueous extract, some of which have been iso-
lated and identified (Ikan and Ravid, 1971; Khafagy et al.,
1979; Sabri et al., 1981; Ghedira et al., 1991; Wessner et al.,
1992; Ben Jannet et al., 1997; and the references quoted
earlier). Since, phytotoxicity usually exists at the level of
the genus, the toxic effects observed may also be due to yet
unidentified additional phytochemicals in AI, which have
been isolated from other plants of genus Ajuga (Camps et al.,
1987; Shimomura et al., 1987; Shen et al., 1993; Beauchamp
et al., 1996; Chen et al., 1996; Terahara et al., 1996;
Bremner et al., 1998; Malakov and Papanov, 1998; Takasaki
et al., 1998; Ben Jannet et al., 1999; Cantrell et al., 1999;
Khan et al., 1999; Nawaz et al., 1999; Fujimoto et al., 2000;
Ben Jannet et al., 2000; Konoshima et al., 2000; Terahara
et al., 2001).
In contrast to the observed toxicity of the intraperitoneal
injections, the lack of adverse effects after oral adminis-
tration of the AI-extract in mice may be explained by low
bioavailability of the “toxic component(s)” due to poor ab-
sorption from the gastrointestinal tract or as a result of high
first-pass effect and rapid metabolism to non-toxic metabo-
lites.
Nevertheless, since the LD50 value for the intraperitoneal
dose is also relatively high, and human exposure to crude
extracts of whole Ajuga iva plant is very unlikely to occur
by the parenteral route, it can be concluded that AI is non-
toxic.
In the chronic toxicity study in rats given the AI-extract
orally at doses of 100–600 mg/kg, there was no change in
animal behaviour, and the body weight gains were not sig-
nificantly different in the treated rats (Groups II–IV) as com-
pared to the controls (Group I). Since, the changes in body
weight have been used as an indicator of adverse effects of
drugs and chemicals (Tofovic and Jackson, 1999; Raza et al.,
2002; Teo et al., 2002), the present results suggest that at the
oral doses administered, the AI-extract is non-toxic in rats.
With the exception of a transient increase in platelet
counts and a temporary decrease in blood glucose, there
were no significant alterations in the haematological and
biochemical parameters of rats. Since there was no effect
on the levels of transaminases (ALT, AST) and creatinine,
which are good indicators of liver and kidney functions,
respectively, it is reasonable to deduce that the AI-extract
did not induce any damage to the liver and the kidneys.
This is further confirmed by the histological assessment of
48 J.E. Hilaly et al. / Journal of Ethnopharmacology 91 (2004) 43–50
these organs (see later), and the fact that there was no effect
on plasma cholesterol levels, the latter being an indirect
indicator of liver function.
The decrease in blood glucose of normal rats confirms
our previous finding that the plant extract acts as a hy-
poglycaemic drug. In the previous study, the plant extract
(at a low oral dose of 10 mg/kg) acted rapidly to decrease
blood glucose both in normoglycaemic and hyperglycaemic
rats, but there was no sustained hypoglycaemia until after
21 days of continuous daily treatment (Hilaly and Lyoussi,
2002). In the present study, hypoglycaemia was observed
for at least 30–60 days after daily dosing with higher doses
(100–600 mg/kg) of the AI-extract. This decrease in blood
glucose is due to an extra-pancreatic action, since plasma in-
sulin levels remained unchanged with the treatment (Hilaly
and Lyoussi, 2002). It is surprising that the decrease in blood
glucose levels in rats was not excessive, considering the
high doses administered, which were 10- to 60-fold higher
than the effective dose (Hilaly and Lyoussi, 2002). It is pos-
sible that there is an absorption threshold (an upper limit)
from the gastrointestinal tract for the active hypoglycaemic
component(s) of the AI-extract. The subsequent loss of hy-
poglycaemic activity on day 90 with repeated dosing with
AI-extract may be due to the homeostasis mechanism of
blood glucose regulation in normal rats.
Histopathological examination of selected organs (brain,
liver and kidney) recovered from treated and control an-
imals showed normal architecture (data not presented),
suggesting no detrimental changes and morphological dis-
turbances were caused by the daily oral administration of
the AI-extract for 90 days.
To confirm the non-toxic nature of any plant product, one
has to consider several factors that can alter its toxicity pro-
file, including the growth stage and maturity of the plant, the
specific part(s) of the plant (such as leaves, roots, bark, flow-
ers, seeds, etc.) used, the seasonal variation in the relative
abundance of phytochemicals, the storage conditions of the
product (freshly collected or stored for a long time), etc. To
balance the effects of these variables, studies should be con-
ducted with the crude plant extract made with a mixture of
freshly harvested whole plants of different ages and growth
stages, collected at various times of the year. Further, since
it is well-known that there is no consistent relationship be-
tween single dose LD50 and daily dosing (Zbenden, 1963),
additional long-term studies with graded doses of AI-extract
are needed to rule out any long-term adverse effects.
In conclusion, at the oral doses tested, AI can be consid-
ered safe as it did not cause either any lethality or adverse
changes in the general behaviour in the acute toxicity stud-
ies in mice and rats, and in chronic toxicity studies in
rats. In normoglycaemic humans, the plant may not pose
a danger in regards to hypoglycaemia, since homeosta-
sis mechanisms may attenuate the initial hypoglycaemic
response. Additional studies in hyperglycaemic rats are
needed to determine the long-term efficacy of AI-extract as
a hypoglycaemic agent.
Acknowledgements
The authors are indebted to Drs. Nicole Morel and Cather-
ine Godfraind, Faculty of Medicine, UCL, Belgium, for their
help with examination of histological cuts.
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